Synthesis of new tetrazolyldienylphenothiazines as potential multidrug resistance inhibitory compounds

Ring opening of tetrazolopyridinium salts with phenothiazines yielded stable cis-trans tetrazolyldienamines. One of the new derivatives proved to inhibit significantly the multidrug resistant efflux pump mechanism and, thus, these new phenothiazine derivatives are regarded as promising candidates for further drug research in the MDR area.


Introduction
Cancer is the third or second most important cause of death worldwide.Multidrug resistant (MDR) efflux pumps extrude anticancer drugs from the tumor cells and, therefore, multidrug resistance leads to treatment difficulties in the majority of cancer patients.The effectiveness of chemotherapy could be increased by the combination of cytostatic drugs with resistance modifiers.Compounds that inhibit the activity of ATP-binding cassette (ABC) transporters can enhance drug accumulation in cancer cells and, consequently, the antiproliferative or cytotoxic action for tumor cells could be enhanced.Several years ago some amphiphilic compounds such as traditional phenothiazines were found to be effective inhibitors of MDR1 encoded phosphoglycoprotein 170 KD (pgp) efflux protein in a relatively high concentration in vitro.1-3  Based on the former results the main aim of our study was to develop new and more effective phenothiazines with MDR reversal action.
In the course of our earlier results on fused azolium salts we elaborated a facile synthetic route to various azolyl dienamines [4][5][6][7] and we have found that the phenothiazine moiety also can be introduced into these compounds. 8These derivatives can be obtained in a single step by ring opening of azolopyridinium salts (2) by secondary amines.Thus, a series of tetrazolyldienamines has been obtained.
Based on some recent biological results in the area of efflux pump inhibitory compounds, 1 an enhanced interest has been focused to phenothiazine derivatives, particularly to those having a carbon chain substituent on the nitrogen atom.This recognition prompted us to apply our preparative technique for the synthesis of novel phenothiazines having a dienyl chain attached to a substituted tetrazole ring.
To this end, we decided to extend our well established ring opening methodology for the synthesis of phenothiazinyldienes and, in light of the variation possibilities in the structure, introduction of different functional groups seemed to be straightforward.In this paper, we describe the synthesis of seven differently substituted phenothiazinyldienes (4a-g) by ring opening of the tetrazolopyridinium salts (2a-c), easily accessible by cyclization of triazenes 1a-c.
Transformation of 2 to 4 was carried out by treatment of the starting material with the sodium salt of the appropriate phenothiazine (3) at room temperature in tetrahydrofuran.Comparison of the coupling constants in the 1 H-NMR data of the dienyl products to those observed with analogous compounds prepared earlier 8 revealed that the double bond C3-C4 retained its cis geometry, whereas the C1-C2 double bond had a trans substitution pattern.
In order to test the biological activity of the new compounds, one representative derivative: 4e was subjected to investigations and its reversal of MDR was studied in human MDR1 gene transfected mouse lymphoma cells by measuring rhodamine 123 accumulation in the model experiments.The efficiency of this new phenothiazine (4e) seems to be promising because 4 µg/ml of 4e increased the rhodamine accumulation by 2.5 fold in our in vitro flow cytometric studies.

Samples
Further structural modifications of phenothiazines of this type and tests of the new derivatives are in progress.

Conclusions
These results indicate that the preparative pathway elaborated by us to the new dienylphenothiazines may open a relatively facile route to a new group of efflux pump inhibitory compounds.Further investigations in this area are in progress.

Experimental Section
General Procedures.Melting points were determined using a Büchi apparatus and are uncorrected.The IR spectra were recorded with an Avatar 320 FT-IR spectrophotometer; the 1 H and 13 C NMR spectra were recorded using Varian UNITY-INOVA (400 MHz) and Varian Gemini 2000 (200 MHz) spectrometers.Starting materials 1a,b and 2a,b were prepared according to the literature procedure. 9

General procedure for the synthesis of butadienylphenothiazines
Sodium hydride (60% suspension in paraffin, 0.046 g, 1.21 mmol) was added to freshly distilled tetrahydrofuran (10 ml) under water-free conditions and the appropriate phenothiazine (1.1 mmol) was added.The mixture was stirred for 10 min at room temperature.Then, the tetrazolopyridinium salt (2, 1 mmol) was added to the reaction mixture in small portions in 5 min, approximately, and stirring was continued for an additional 60 min.The yellow colored suspension turned dark.The reaction mixture was poured onto ice-water and the product was extracted with dichloromethane (3 x 20 ml), the combined organic layer was washed with water and was dried over sodium sulfate.After evaporation the residue was triturated with diethyl ether -unless otherwise mentioned -to yield the pure product.Samples for analysis were either recrystallized from the given solvent or -if decomposition took place upon heating -were further purified by chromatography.